Grapevine trunk diseases are responsible for significant economic
losses to the wine industry worldwide. Symptoms of these diseases
include dead spurs, arms, and cordons and eventual vine death due
to canker formation in the vascular tissue. In Eutypa dieback, deformed
leaves and shoots occur as the pathogen invades spur positions.
As cankers develop, yield reductions occur due to the loss of productive
wood. The impact of grapevine wood diseases can be significant in
older vineyards, and usually becomes more severe as vineyards become
older.

Eutypa dieback, caused by Eutypa lata was originally thought to
be responsible for most canker development in California vineyards.
However, recent findings have highlighted the importance of other
fungi involved in the death and decline of grapevines in California.
In this regard, Botryosphaeria species have also been recovered
from cankers, and were determined to be the main cause of canker
diseases in some California vineyards.

Recent research has also indicated the occurrence of several new
fungal trunk disease pathogens of grapevine belonging to the family
Diatrypaceae (the same family as Eutypa). These include Eutypa leptoplaca,
Cryptovalsa ampelina, Diatrype species, and Diatrypella species.
We will present current information on the epidemiology and control
strategies of fungal organisms responsible for grapevine spur, cordon,
and trunk dieback in California.

Eutypa dieback
Eutypa dieback was responsible for a loss in net income for California
wine grapes estimated to be over $260 million in 1999. Many growers
consider Eutypa to be the most significant disease of grapevines.

Typical symptoms of E. lata include formation of a wedge-shaped
canker and stunted shoots with cupped, tattered, chlorotic, and
necrotic leaves that are best seen in spring time. Foliar symptoms
are due to toxins produced by E. lata. Differences in susceptibility
of grapevine cultivars to infection have been reported, although
no cultivars are immune. Cankers develop downward at a faster rate
than toward the end of cordons and also increase in diameter over
time. Extended infection of grapevines by E. lata leads to vine
death.

Eutypa
lata spreads to new pruning wounds by wind-driven and water-splashed
ascospores released during rain events. Ascospores develop inside
perithecia (fungal fruiting bodies) that form when the fungus enters
its sexual stage. The sexual stage develops on dead wood, where
masses of perithecia are produced in a black substrate referred
to as stroma (Figure II). The sexual stage develops in regions that
receive over 16 inches of rain. It is common to find stroma and
perithecia on old grapevines and other types of wood in the North
Coast and Delta production areas.

Eutypa lata also produces asexual spores called conidia. These are
formed inside pycnidia (another type of fruiting body) that develop
on wood, but these conidial spores do not play a role in disease
epidemiology.

In California, ascospore discharge of E. lata occurs
from the first rain of the early fall until the last rains of the
spring (Figure III). Ascospore discharge decreases significantly
in late February and remains low to nil by early March (Figure IX).

However,
ascospore release may occur during rains in March and April if they
are preceded by several weeks of no rain and sunny, warm weather.
Such releases may occur because perithecia are able to recover in
productivity during the dry period, or because spores that would
have been released in the winter months are released in the spring
simply because they were not released in the winter. This scenario
more often occurs in years when there is little rainfall during
the winter months.

Ascospore release from individual perithecia may occur continuously
for approximately 24 hours during periods of rainfall, starting
a few hours after the onset of a rain (Figure IX).

Over 80 plant species around the world have been reported to be
potential hosts for E. lata. In California, many of these species
were found to be infected with Eutypa lata and bore perithecia producing
ascospores in the vicinity of vineyards. We now know that these
species serve as natural reservoirs of E. lata inoculum.

In the 1970s, grapevine, apricot, and Ceanothus were found to be
natural hosts of E. lata. More recently we have identified kiwifruit,
blueberry, and cherry to be hosts in California and they were shown
to bear the fruiting bodies (perithecia) of E. lata.

Additionally, a recent survey identified almond, crab apple, and
pear trees as new fruit crop hosts for E. lata, and showed the presence
of perithecia of E. lata on several other new hosts in California
including California buckeye, big leaf maple, willows, and oleander
(Figure X).

Perithecia of E. lata were found to be particularly well established
on dead branches of various willow species occurring along natural
creeks and irrigation waterways.

It
appears likely that the flora surrounding vineyards is potentially
a key factor in disease epidemiology and surely acts as an inoculum
reservoir. Sanitation of the dead wood of potential hosts of E.
lata in areas surrounding vineyards is advised in order to decrease
the inoculum level. Sanitation can be accomplished by removing the
infected tissues on plants that show disease. Generally speaking,
this disease can be identified on other hosts by the black stroma
that is produced (Figure II). By removing the stroma-infected wood
and burning it, the number of spores released in winter can be reduced.

Surveys inside vineyards and apricot and cherry orchards have revealed
an abundance of inoculum in plantings of approximately 20 years
and older. Only a few perithecia have been found in almond orchards.

Perithecia of E. lata were found to be prevalent in vineyards or
surrounds in the counties of Napa, Sonoma, Yolo, Sacramento, Contra
Costa, San Benito, El Dorado, Mendocino, San Joaquin, Stanislaus,
and Merced. Perithecia of E. lata were not found in Madera, Fresno,
Kings, Tulare, and Kern counties.

Large amounts of viable inoculum were found in several old vineyards
near Healdsburg, Sonoma County. Perithecia were particularly well-developed
on vines that had been previously top-worked for variety change.
Stroma on those vines had developed on old wood below the grafting
wound down to the union with the rootstock (Figure I).

While E. lata appears to be the primary Eutypa species involved
in dieback in California, we have identified a second Eutypa species
that can also cause dieback in grapevines (Figure XIII). This species
is E. leptoplaca, a slower growing fungus in culture but with the
capability to do the same kind of damage as E. lata. This new fungus
occurs in the North Coast but seems to be more limited in its distribution.

It is our opinion after this study that even though E. lata and
E. leptoplaca ascospores can travel over considerable distances
and cause disease, Eutypa dieback is primarily a disease of local
origin, developing in the vicinity where the ascospores are released.

Control
Disease management should include removal of the dead parts of grapevines
and other host plants because of the potential risk of increased
inoculum. We currently have no proof that sanitation will reduce
the disease level in vineyards, but it seems appropriate if local
production of inoculum is considered to be a key to disease development.

Grapevine pruning wounds can be susceptible to infection by E. lata
for as long as seven weeks, but the length of this period varies
with the time of pruning, size of the wound, and age of the wood
pruned. Wound susceptibility to E. lata declined faster under higher
(70† to 90†F) temperatures.

Wound healing occurs by deposition of polymerized phenolic compounds
in the opened wood vessels, and concomitantly, natural establishment
of a microbial population on the wound surface provide a natural
bio-barrier to infection. These natural organisms (epiphytes),
including Cladosporium herbarum and Fusarium lateritium, grow over
the surface of a pruning wound and prevent infection by E. lata
spores.

In a previous study, more than 1300 species of fungi and bacteria
were found to colonize grapevine wounds in spring conditions.

Wound healing and colonization by epiphytic fungi and bacteria have
been shown to occur more readily in the warmer spring months. Ascospore
release, germination, and infection are also decreased during this
time. Therefore, significant wound protection can be readily achieved
simply by late pruning of vineyards.

Our work, and other research (Moller and Kasimatis), has shown that
pruning wounds made in March heal more rapidly when compared to
mid-winter. In our trials, late season pruning wounds that were
artificially inoculated showed only approximately 10% of the infection
rate of pruning wounds inoculated in mid-winter.

However, the large acreage of individual vineyards in California
makes late pruning impractical for many growers who need to schedule
their labor force throughout the winter months. One way to allow
for more efficient late season pruning is to double-prune a vineyard.
This involves going through the vineyard twice during the pruning
season.

The first pre-pruning pass is made early in the season
at which time the canes are simply trimmed back to a set length,
usually 12 to 18 inches. This step can be performed mechanically
or by a hand crew. Final pruning to spurs would take
place late in the season (March) when the chances for infection
are reduced.

By eliminating most of the brush early in the winter,
final pruning should go more quickly, thereby allowing the final
cuts to be made late in the season. Double-pruning is not appropriate
for cane-pruned vineyards where long canes are retained each year.

Double-pruning creates an opportunity for wood pathogens such as
E. lata to colonize wounds made early in the dormant season during
pre-pruning. However, these infections will be eliminated during
the final pruning step. We inoculated pre-pruning cuts throughout
the winter months and showed that by March, E. lata had not grown
downward more than four to five cm, which was well above the point
where final pruning cuts were made.

Late pruning, either in a single step or as the final
pass in double-pruned vineyards, is not a guarantee that Eutypa
infections will not occur. Late spring rains can trigger ascospore
releases and increase the risk of infection of newly pruned vineyards.
However, late pruning certainly reduces the overall chances for
infection and is a standard recommendation for disease control.

Disease management was historically achieved by treating pruning
wounds with fungicides. Benlate® (DuPont de Nemours & Co.,
U.S.) was registered for E. lata for 30 years, and field trials
showed good efficacy in preventing Eutypa dieback. However, long-term
protection of pruning wounds was not achievable because the product
did not persist in woody tissue for a sufficient time period. Therefore,
multiple applications of Benlate would have been required to provide
protection until pruning wounds were completely healed.

Benlate was withdrawn from the market in 2001 leaving
growers without an efficacious chemical treatment to control E.
lata. Topsin M was registered in 2003 under section 18 (emergency
exemption) in California, but this product also belongs to the same
class of fungicides as Benlate, a benzimidazole, and therefore has
the same drawback (short-term protection) as Benlate.

Biocontrol agents have been tested as an alternative method for
control of E. lata. Bacillus subtilis, Fusarium lateritium, and
Cladosporium herbarum all showed some potential activity in limiting
the establishment of the pathogen.

However, unlike chemical applications, which have an immediate protective
effect, maximum protection from biocontrol agents requires colonization
of the surface of the wound. Thus, there is a window of susceptibility
after treatment, until the biocontrol agent is established well
enough to prevent development of E. lata in the wounded tissue.

Biocontrols tested as alternatives to fungicides showed
mixed success, but both F. lateritium and C. herbarum worked well
when they were applied two to three weeks before infection occurred.

Boron was tested as an alternative control method because this element
was proven to be effective at controlling other wood decay fungi.
It is also used by growers as a fertilizer spray to improve fruit
set. The application of boron as a fungicide may not yet be legal
to control Eutypa dieback of grapevines, however, boron can be applied
to correct nutritional problems.

Boric acid reduced ascospore germination and mycelial
growth of E. lata under controlled conditions in the laboratory,
and the boron-based products yielded excellent disease control on
artificially inoculated grapevines in both field trials and lab
studies in comparison to C. herbarum and boron-free paste treatments
(Table I).

However, in one observation, the bud located at the first node below
the pruning wound failed to push following treatment with liquid
boric acid. This effect was also reported in peach and nectarine
orchards sprayed with toxic amounts of boron-based fertilizers.
Mechanisms involved in bud failure are not understood, and the economic
impact has not been assessed. However, we do not recommend liquid
boric acid for Eutypa dieback control.
Treatment of pruning wounds with boron-based products offers an
effective, economical, and environmentally-safe management strategy
to control Eutypa dieback in vineyards. However, formulations have
to be optimized in order to increase control over longer time periods
on the surface of pruning wounds, and to limit possible effects
on bud failure of grapevines.

We believe strongly that if boron is used, it should
be put in a paste so that it stays where it is placed.

In earlier studies, soaps also provided good protection of pruning
wounds against E. lata. However, most soaps, including dishwashing
detergents, were phytotoxic. One laundry detergent, Dreft, not only
provided excellent control of E. lata, but has never shown any phytotoxicity.
This detergent, when used at 30% aqueous suspension (wt/vol), provided
excellent disease control.

Further fungicide testing is underway, and we are trying to convince
the chemical industry that Eutypa control products are worth registering
for California viticulture.

BOTRYOSPHAERIA canker diseases
Botryosphaeria canker disease of grapevine is a wood
disease caused by as many as four different Botryosphaeria species
in California. Although it is well accepted that some Botryosphaeria
species have been shown to be the causal agent of canker diseases
of various crops and woody plants in California, the importance
of these fungi as pathogens of grapevines has been largely ignored
throughout the state.

Various species of Botryosphaeria, causing different symptoms, have
been reported in the last decade as pathogens on grapevines in the
U.S., South Africa, Australia, France, Italy, Portugal, Egypt, India,
Mexico, Chile, and Brazil. In California, wedge-shaped cankers caused
by Botryosphaeria can be found on vines 10 years old and older,
especially where large pruning wounds have been made in retraining
vines.

For several years, Botryosphaeria rhodina has been known to cause
wedge-shaped canker symptoms in California. This species was previously
known as Botryodiplodia theobromae, and the disease it causes was
referred to as Bot canker.

Fungi often produce both an asexual (imperfect) stage
and a sexual (perfect) stage, and they may have different names.
Botrysphaeria rhodina is the name for the sexual stage of this fungus;
Botryodiplodia theobromae is the name of the asexual stage.

We prefer to use the name Botryosphaeria instead of Botryodiplodia.
The disease is still known as Bot Canker. The fungus is now considered
to be an endemic species in many vineyards in warm and hot climate
areas in California.

Typical symptoms caused by Botryosphaeria on grapevines in California
are wedge-shaped cankers in the trunk and cordons (Figure IV) and
dead spur positions. No foliar symptoms associated with Botryosphaeria-induced
canker diseases have been observed in California grapevines. This
is in contrast to other areas in the world where foliar symptoms
have been observed on grapevines infected by different species of
Botryosphaeria (B. stevensii, B. obtusa and B. dothidea).

The wedge-shape cankers caused by Botryosphaeria are
visually indistinguishable from those formed by Eutypa lata or E.
leptoplaca. All three fungi can cause cankers and can be detected
in the vine at the same time. The best distinguishing characteristic
is the presence of cankers and the absence of the stunted or chlorotic
spring growth which is typical of infections by Eutypa lata.

Botryosphaeria spp. are wound pathogens entering the vine through
fresh pruning wounds. Large numbers of Botryosphaeria conidia are
exuded from black fruiting bodies (pycnidia) found on diseased vine
parts, under the bark of cordons, trunks, and spurs (Figure VII)
or on the residual pruning wood left in the vineyards.

In addition, we also know that many of these species
cause disease on different hosts around the vineyards. The formation
of numerous fruiting bodies provides an excellent source of spores
for further infections in the vineyard. Conidia may be easily distributed
over the vineyard due to wind, or they may be waterborne in splashed
drops from rain or sprinkler irrigation, but very little information
is available concerning the Botryosphaeria disease cycle.

Like Eutypa lata and E. leptoplaca, the canker formed by Botryosphaeria
spp. grows more rapidly basipetaly (or toward the root from the
point of infection). The canker develops for several years in the
trunks and cordons depending on where the infection was located.
Death of the infected vine part occurs when the last live wedge
of tissue is killed by the growth of the fungus.

Eutypa dieback and Bot canker disease decrease the life
of the vineyard, reduce yields and increase production costs due
to the application of control treatments, cultural practices to
prevent infections, pruning out of diseased tissue, and training
new cordons and spur positions to replace those killed by the disease.

Other wood decay fungi
Besides Eutypa lata and Botryosphaeria species, several other fungi
were recovered from cankers of grapevines. These include Phomopsis
viticola, Phaeomoniella chlamydospora, and Phaeoacremonium aleophilum.
Others, such as Diatrypella sp., Diatrype sp., Crytovalsa ampelina
and E. leptoplaca were also isolated from the margins of infected
tissue.

Recently, another Eutypa species (E. leptoplaca) and Cryptovalsa
ampelina were found to be pathogenic on grapevines as were species
of Diatrypella and Diatrype. These fungal species all belong to
the same family as E. lata and closely resemble E. lata in their
spore shape, size, and by their appearance in culture (Figure XIII).
These fungi also have been found commonly on various host plants
in the vicinity of vineyards.

Eutypa lata and the other diatrypaceous fungi have
the same morphological characteristics in culture including spore
shape and color. It is apparent that over the last several years,
E. lata was not the only pathogen being isolated from cankers. Not
until this study did we realize that more than one pathogen was
capable of causing the same type of disease.

Given these results, it is evident that Eutypa lata is not the only
cause of grapevine canker diseases in California vineyards. More
studies should be conducted to elucidate the impact of the different
species of Botryosphaeria, and other fungi detected in cankers.
The virulence of many of these species remains to be determined.

These recent discoveries have led to the conclusion that grapevine
trunk diseases are more complicated than initially thought, and
that a complex of fungi may be involved. Development of information
regarding the biology, epidemiology and control of each of these
fungi and diseases is underway. Nevertheless, we know that vineyard
sanitation through the removal of infected parts of vines is highly
advised, and where possible, sanitation of surrounding areas where
other potential hosts of these pathogenic fungi reside.

Acknowledgements: We wish to thank
the American Vineyard Foundation and the USDA Viticulture Consortium
for partial funding of this work.